sermorelin and Disease-Models--Animal

Ocular inflammation is a major cause of visual impairment attributed to dysregulation of the immune system. Previously, we have shown that the receptor for growth-hormone-releasing hormone (GHRH-R) affects multiple inflammatory processes. To clarify the pathological roles of GHRH-R in acute ocular inflammation, we investigated the inflammatory cascades mediated by this receptor. In human ciliary epithelial cells, the NF-κB subunit p65 was phosphorylated in response to stimulation with lipopolysaccharide (LPS), resulting in transcriptional up-regulation of GHRH-R. Bioinformatics analysis and coimmunoprecipitation showed that GHRH-R had a direct interaction with JAK2. JAK2, but not JAK1, JAK3, and TYK2, was elevated in ciliary body and iris after treatment with LPS in a rat model of endotoxin-induced uveitis. This elevation augmented the phosphorylation of STAT3 and production of proinflammatory factors, including IL-6, IL-17A, COX2, and iNOS. In explants of iris and ciliary body, the GHRH-R antagonist, MIA-602, suppressed phosphorylation of STAT3 and attenuated expression of downstream proinflammatory factors after LPS treatment. A similar suppression of STAT3 phosphorylation was observed in human ciliary epithelial cells. In vivo studies showed that blocking of the GHRH-R/JAK2/STAT3 axis with the JAK inhibitor Ruxolitinib alleviated partially the LPS-induced acute ocular inflammation by reducing inflammatory cells and protein leakage in the aqueous humor and by repressing expression of STAT3 target genes in rat ciliary body and iris and in human ciliary epithelial cells. Our findings indicate a functional role of the GHRH-R/JAK2/STAT3-signaling axis in acute anterior uveitis and suggest a therapeutic strategy based on treatment with antagonists targeting this signaling pathway.

Topics: Animals; Cell Line; Ciliary Body; Disease Models, Animal; Epithelial Cells; Humans; Janus Kinase 2; Lipopolysaccharides; Male; Nitriles; Pyrazoles; Pyrimidines; Rats; Receptors, Neuropeptide; Receptors, Pituitary Hormone-Regulating Hormone; Sermorelin; Signal Transduction; STAT3 Transcription Factor; Uveitis

Growth hormone-releasing hormone (GHRH) is a 44-amino acid peptide that regulates growth hormone (GH) secretion. We hypothesized that a GHRH receptor (GHRH-R) antagonist, MIA-602, would inhibit bleomycin-induced lung inflammation and/or fibrosis in C57Bl/6J mice.. We tested whether MIA-602 (5 μg or vehicle given subcutaneously [SC] on days 1-21) would decrease lung inflammation (at day 14) and/or fibrosis (at day 28) in mice treated with intraperitoneal (IP) bleomycin (0.8 units on days 1, 3, 7, 10, 14, and 21). Bleomycin resulted in inflammation and fibrosis around airways and vessels evident histologically at days 14 and 28.. Inflammation (histopathologic scores assessed blindly) was visibly less evident in mice treated with MIA-602 for 14 days. After 28 days, lung hydroxyproline (HP) content increased significantly in mice treated with vehicle; in contrast, lung HP did not increase significantly compared to naïve controls in mice treated with GHRH-R antagonist. GHRH-R antagonist increased basal and maximal oxygen consumption of cultured lung fibroblasts. Multiple genes related to chemotaxis, IL-1, chemokines, regulation of inflammation, and extracellular signal-regulated kinases (ERK) were upregulated in lungs of mice treated with bleomycin and MIA-602. MIA-602 also prominently suppressed multiple genes related to the cellular immune response including those for T-cell differentiation, receptor signaling, activation, and cytokine production.. MIA-602 reduced lung inflammation and fibrosis due to bleomycin. Multiple genes related to immune response and T-cell functions were downregulated, supporting the view that MIA-602 can modulate the cellular immune response to bleomycin lung injury.

Topics: Animals; Bleomycin; Cells, Cultured; Cytoprotection; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Gene Expression Regulation; Growth Hormone-Releasing Hormone; Hormone Antagonists; Hydroxyproline; Inflammation Mediators; Lung; Male; Mice, Inbred C57BL; Pneumonia; Pulmonary Fibrosis; Sermorelin; Signal Transduction

We investigated the effects of novel antagonists of growth hormone releasing hormone (GHRH)-MIA602 and MIA690-on three human small cell lung cancer (SCLC) lines (H446, DMS53 and H69) and two non-SCLC (NSCLC) lines (HCC827 and H460). In vitro exposure of cancer cells to these GHRH antagonists significantly inhibited cell viability, increased cell apoptosis, decrease cellular levels of cAMP and reduced cell migration. In vivo, the antagonists strongly inhibited tumor growth in xenografted nude mice models. Subcutaneous administration of MIA602 at the dose of 5 μg/day for 4-8 weeks reduced the growth of HCC827, H460 and H446 tumors by 69.9%, 68.3% and 53.4%, respectively, while MIA690 caused a reduction of 76.8%, 58.3% and 54.9%, respectively. Western blot and qRT-PCR analyses demonstrated a downregulation of expression of the pituitary-type GHRH-R and its splice-variant, cyclinD1/2, cyclin-dependent kinase4/6, p21-activated kinase-1, phosphorylation of activator of transcription 3 and cAMP response element binding protein; and an upregulation of expression of E-cadherin, β-catenin and P27

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Disease Models, Animal; Female; Gene Expression; Growth Hormone-Releasing Hormone; Humans; Mice; Mice, Nude; Sermorelin; Signal Transduction; Small Cell Lung Carcinoma; STAT3 Transcription Factor; Xenograft Model Antitumor Assays

Endometriosis is a benign gynecologic disorder causing dysmenorrhea, pelvic pain, and subfertility. Receptors for the growth hormone-releasing hormone (GHRH) were found in endometriotic tissues. Antagonists of GHRH have been used to inhibit the growth of endometriotic endometrial stromal cells. In this study, the GHRH receptor splice variant (SV) 1 was detected in human endometrial tissue samples by Western blots and quantitative reverse transcription polymerase chain reaction (qRT-PCR). The highest messenger RNA (mRNA) and protein levels of SV1 were found in eutopic endometrium from patients with endometriosis compared to ectopic endometriotic tissues and endometrium from normal patients. The highest expression for GHRH mRNA was found by qRT-PCR in ectopic endometriosis lesions. In an in vivo mouse model with human endometrial explants from patients with endometriosis, 10 μg MIA-602 per day resulted in significantly smaller human endometrial xenotransplants after 4 weeks compared to mice treated with vehicle. The endometrial tissues expressed SV1 before and after xenotransplantation. The proliferation of endometrial stromal cells as well as the endometriosis cell lines 12-Z and 49-Z was decreased by exposure to 1 μM MIA-602 after 72 hours. The protein levels of epithelial growth factor receptors in 12-Z and 49-Z cell lines were reduced 48 and 72 hours after the administration of 1 μM MIA-602. MIA-602 decreased the activation of the MAP-kinases ERK-1/2. Our study demonstrates the presence of SV1 receptor as a target for treatment with GHRH antagonist in endometriosis. Endometrial tissues respond to MIA-602 with inhibition of proliferation in vitro and in vivo. The use of MIA-602 could be an effective supplement to the treatment strategies in endometriosis.

Topics: Adult; Animals; Cell Proliferation; Disease Models, Animal; Endometriosis; Female; Growth Hormone-Releasing Hormone; Humans; Mice; Mice, Nude; Middle Aged; Sermorelin; Transplantation, Heterologous

MZ-4-71 is an antagonist of growth hormone-releasing hormone (GH-RH) which suppresses the secretion of GH-RH. It has been shown that MZ-4-71 has antidepressive-like effects in a modified forced swimming test (FST) in mice, exerts anxiolytic effects in an elevated plus maze test, improves memory consolidation in passive avoidance learning, and corrects the impairment of memory consolidation caused by β-amyloid 25-35 in mice. However, little is known about the mechanisms of action of MZ-4-71 on brain functions. The involvement of the adrenergic, serotonergic, cholinergic, dopaminergic or GABA-ergic receptors in the antidepressant-like action of MZ-4-71 (1.0 μg/2 μl, intracerebroventricular (i.c.v.)) was studied in a modified mouse forced swimming test (FST). Mice were pretreated with a non-selective α-adrenergic receptor antagonist, phenoxybenzamine, an α1/α2β-adrenergic receptor antagonist prazosin, an α2-adrenergic receptor antagonist, yohimbine, a β-adrenergic receptor antagonist, propranolol, a mixed 5-HT1/5-HT2 serotonergic receptor antagonist methysergide, a non-selective 5-HT2 serotonergic receptor antagonist, cyproheptadine, a non-selective muscarinic acetylcholine receptor antagonist, atropine, a D2, D3, D4 dopamine receptor antagonist, haloperidol or a γ-aminobutyric acid subunit A (GABA-A) receptor antagonist bicuculline. Phenoxybenzamine, prazosin, methysergide, cyproheptadine and atropine prevented the effects of MZ-4-71 on the immobility, the climbing and the swimming times. Yohimbine, propranolol, haloperidol and bicuculline did not change the effects of MZ-4-71. The results demonstrated that the antidepressant-like effects of MZ-4-71 in this modified mouse FST are mediated, at least in part, by the an interaction of the α1-adrenergic, 5-HT1/5-HT2 serotonergic, and muscarinic acetylcholine receptors.

Topics: Analysis of Variance; Animals; Antidepressive Agents; Depression; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Growth Hormone-Releasing Hormone; Immobility Response, Tonic; Injections, Intraventricular; Male; Mice; Muscle Strength; Neurotransmitter Agents; Sermorelin; Swimming

Antagonists of growth hormone-releasing hormone (GH-RH), such as MZ-4-71 suppress the secretion of GH. These findings suggest that GH-RH antagonists could be used for the therapy of disorders characterized by excessive GH secretion. It has been also demonstrated that MZ-4-71 displays antidepressant effects in a modified forced swimming test in mice, exerts anxiolytic effects in an elevated plus maze test, improves memory consolidation in passive avoidance learning, and corrects the impairment of memory consolidation caused by β-amyloid (25-35) in mice. However, little is known about the mechanisms of action of MZ-4-71 on brain functions. In the present work, the involvement of the adrenergic, serotonergic and GABA-ergic receptors in the anxiolytic action of MZ-4-71 was studied in an elevated plus maze. Mice were pretreated with a nonselective α-adrenergic receptor antagonist, phenoxybenzamine, an α1/α2β-adrenergic receptor antagonist, prazosin, an α2-adrenergic receptor antagonist, yohimbine, a mixed 5-HT1/5-HT2 serotonergic receptor antagonist, methysergide, a non-selective 5-HT2 serotonergic receptor antagonist, cyproheptadine, and a γ-aminobutyric acid subunit (GABA-A) receptor antagonist, bicuculline. Phenoxybenzamine, prazosin, yohimbine, methysergide, cyproheptadine and bicuculline prevented the effects of MZ-4-71 on the elevated plus maze revealing that the anxiolytic actions of MZ-4-71 in this test are mediated, at least in part, by the an interaction of the α1/α2-adrenergic, 5-HT1/5-HT2 serotonergic and GABA-A-ergic receptors.

Topics: Analysis of Variance; Animals; Anti-Anxiety Agents; Anxiety; Avoidance Learning; Bicuculline; Cyproheptadine; Disease Models, Animal; Growth Hormone; Growth Hormone-Releasing Hormone; Male; Maze Learning; Methysergide; Mice; Neurotransmitter Agents; Phenoxybenzamine; Prazosin; Sermorelin; Swimming

Since antagonists of growth hormone-releasing hormone (GH-RH) inhibit proliferation of various tumors, in this study we investigated the effects of GH-RH antagonists MZ-5-156 or JV-1-36 on growth of estrogen-independent MDA-MB-468 human breast cancers xenografted into nude mice. Both GH-RH antagonists administered at a dose of 20 microg/day induced regression of some and growth-arrest of other tumors, while control tumors continued to grow. After 5 weeks of therapy with MZ-5-156 or JV-1-36, final volume and weight of MDA-MB-468 tumors were significantly decreased (all p values < 0.001) and serum IGF-I levels as well as tumor IGF-I mRNA expression were reduced as compared with controls. High affinity binding sites for IGF-I were detected by the ligand binding method. Gene expression of human IGF-I receptors, as measured by the RT-PCR, was not significantly different in control and treated MDA-MB-468 tumors. In cell culture, IGF-I did not stimulate, GH-RH slightly stimulated, while MZ-5-156 and JV-1-36 inhibited proliferation of MDA-MB-468 cells known to possess defective insulin and IGF-I receptor signaling. The expression of mRNA for human GH-RH was found in five of 8 tumors treated with GH-RH antagonists, and in one of the five control tumors. These results suggest that GH-RH antagonists inhibit MDA-MB-468 breast cancers possibly through mechanisms involving interference with locally produced GH-RH.

Topics: Animals; Breast Neoplasms; Disease Models, Animal; DNA Primers; Female; Gene Expression Regulation, Neoplastic; Growth Hormone-Releasing Hormone; Humans; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Mice; Mice, Nude; Radioimmunoassay; Receptor, IGF Type 1; Receptors, Neuropeptide; Receptors, Pituitary Hormone-Regulating Hormone; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sermorelin; Transplantation, Heterologous; Tumor Cells, Cultured

Transgenic mice overexpressing the human GH-releasing hormone (hGHRH) gene, an animal model of acromegaly, were used to investigate the effects of potent GHRH antagonists MZ-4-71 and MZ-5-156 on the excessive GH and insulin-like growth factor I (IGF-I) secretion caused by overproduction of hGHRH. Because metallothionein (MT)-GHRH mice express the hGHRH transgene in various tissues, including the pituitary and hypothalamus, initial experiments focused on the effectiveness of the GHRH antagonists in blocking basal and stimulated GH secretion from pituitary cells in vitro. Both MZ-4-71 and MZ-5-156 suppressed basal release of GH from superfused MT-GHRH pituitary cells, apparently by blocking the action of endogenously produced hGHRH. In addition, these antagonists effectively eliminated the response to stimulatory action of exogenous hGHRH(1-29)NH2 (30 and 100 nM). To ascertain whether MZ-4-71 and MZ-5-156 could antagonize the effect of hGHRH hyperstimulation in vivo, each antagonist was administered to MT-GHRH transgenic mice in a single iv dose of 10-200 microg. Both compounds decreased serum GH levels in transgenic mice by 39-72% at 1 h after injection. The inhibitory effect of 50 microg MZ-5-156 was maintained for 5 h. Twice daily ip administration of 100 microg MZ-5-156 for 3 days suppressed the highly elevated serum GH and IGF-I concentrations in transgenic mice by 56.8% and 39.0%, respectively. This treatment also reduced IGF-I messenger RNA levels in the liver by 21.8% but did not affect the level of GH messenger RNA in the pituitary. Our results demonstrate that GHRH antagonists MZ-4-71 and MZ-5-156 can inhibit elevated GH levels caused by overproduction of hGHRH. The suppression of circulating GH concentrations induced by the antagonists seems to be physiologically relevant, because both IGF-I secretion and synthesis also were reduced. Our findings, showing the suppression of GH and IGF-I secretion with GHRH antagonists, suggest that this class of analogs could be used for the diagnosis and therapy of disorders characterized by excessive GHRH secretion.

Topics: Acromegaly; Animals; Disease Models, Animal; Drug Administration Schedule; Female; Gene Expression; Growth Hormone; Growth Hormone-Releasing Hormone; Hormone Antagonists; Humans; Insulin-Like Growth Factor I; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Sermorelin